Small-molecule inhibitors of protein kinases constitute a novel class of drugs for therapeutic intervention in a variety of human diseases. Most of these agents target the relatively conserved ATP-binding site of protein kinases and have only been tested against a rather small subset of all human protein kinases. Therefore, the selectivity of protein kinase inhibitors has remained a widely underestimated, but highly important issue in drug development programs. In this review, we focus on the recent advancement of chemical proteomic methods to evaluate drug selectivity in an unbiased, comprehensive way. Efficient affinity purification procedures using immobilized kinase inhibitors combined with the sensitivity of mass spectrometry detection permit the mapping of drug targets on a proteome-wide scale. Data from this type of assessment can be used to set up tailor-made selectivity panels, which guide compound development in the context of the most relevant off-targets during lead optimization. In cases in which identified alternative targets are of validated clinical relevance, chemical proteomics provides the opportunity to repeatedly exploit a once established kinase inhibitor principle for additional target kinases and can thereby dramatically shorten the time toward highly selective, preclinical candidates. Moreover, the identification of alternative targets for preclinical or clinical drugs can provide new insights into their cellular modes of action, which might help to define those disease settings in which the most beneficial therapeutic effect is likely to occur.